The lack of effective intracellular delivery systems for therapeutic compounds continues to hamper the progress of many types of biological study, and impedes the development of clinical applications of that research. Intracellular delivery of many molecules can be difficult but the delivery of extremely large or complex molecules, such as the DNA that comprises human genes, is particularly problematic. For example, gene delivery has been described as “[T]he Achilles heel of gene therapy”. (Verma, I. M. et al. 1997, Nature 389:239-242, 239) While gene therapy is universally recognized as having tremendous clinical potential, it has yet to achieve completely successful clinical results. (Friedmann, T., 1997, Ann. Med. 6:575-577)
At present, the majority of gene therapy systems rely on viral vectors for gene delivery. Retroviral, adeno-associated and other viral strains have all been utilized as nucleic acid vectors, due to their ability to infect a large proportion of target cells in a manner which results in stable integration of the nucleic acid in the cell. However, such vectors lack the capacity to deliver large DNA molecules and, in addition, difficulties with sustained protein expression in cells subsequent to viral infection have been widely reported. (Hodgson, C. et al., 1996, Retro-Vectors for Human Gene Therapy, Chapter 6, pp. 129-145) Furthermore, there are ever increasing concerns regarding the ability to safely use viral vectors in the clinical setting. (Crystal, R., 1995, Science 170:404-410)
Such concerns have resulted in increased interest in nonviral alternatives for intracellular compound delivery. One major hurdle in developing systems of this type is the inability of certain molecules, such as small molecules, proteins, peptides, oligonucleotides, and genes, to efficiently traverse the lipid bilayer of the cell plasma membrane or that of endosomal vesicles. Certain types of transport systems have been developed to allow compounds to enter cells through receptor mediated endocytosis in an attempt to solve the difficulties of efficient cell entry. These systems contain conjugates formed of a polycation domain which binds the delivery compound, and a ligand domain which targets a receptor for compound delivery. (Harbottle, R. P. et al., 1998, Human Gene Therapy 9:1037-1047; Schaffer, D. V. et al., 1998, J Biol Chem 43:28004-28009) However, the effectiveness of these systems is severely impacted by the degradation to the compounds which occurs in the endosomes following cell entry. (Palliard, F., 1998, Human Gene Therapy 9:987-988)
Another alternative to viral vectors are various cationic lipids, liposomes, that allow genes to cross the cell membrane. In general, the efficiency of delivery and sustained expression provided by these vectors has been poor. (Verma, I., et a. 1997, Nature 389:239-242) Moreover, there have been reports of toxic side effects with some liposomes.
Other systems have recently emerged which attempt to target the lipid bilayer component of the cell directly, and thus avoid the difficulties associated with receptor mediated endocytosis. Such systems generally contain a moiety able to translocate across biological membranes attached to the “cargo” intended for delivery. One such system, containing the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein, has been used to deliver a β-galactosidase protein to cells in an animal model. (Schwarze, S.R et al., 1999, Science 285:1569-1572) Another such system, containing a portion of the homeodomain protein from antennapedia (hereinafter “ANT”), has demonstrated delivery of several small proteins and oligonucleotides to cells in culture. It has been reported, however, that monomeric homeodomains “lose their translocation abilities when they bind to DNA”. (Derossi, D. et al., 1998, Trends in Cell Biology 8:84-87) Thus, the range of compounds which these systems are currently able to deliver is limited in type and size.
Therefore, there exists a need for a versatile system capable of effectively delivering a wide range of therapeutic compounds intracellularly.